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Dive into the research topics where Alexandre V. Machado is active.

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Featured researches published by Alexandre V. Machado.


Infection and Immunity | 2009

Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination

Bruna Cunha de Alencar; Pedro M. Persechini; Filipe A. Haolla; Gabriel de Oliveira; Jaline Coutinho Silverio; Joseli Lannes-Vieira; Alexandre V. Machado; Ricardo T. Gazzinelli; Oscar Bruña-Romero; Mauricio M. Rodrigues

ABSTRACT A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4+ and CD8+ T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4+ and CD8+ T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-γ) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8+ T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-γ or IFN-γ/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-γ in the presence of highly cytotoxic T cells. Vaccinated IFN-γ-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-γ in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy.


Journal of Immunology | 2008

Infection with Trypanosoma cruzi Restricts the Repertoire of Parasite-Specific CD8+ T Cells Leading to Immunodominance

Fanny Tzelepis; Bruna Cunha de Alencar; Marcus L. O. Penido; Carla Claser; Alexandre V. Machado; Oscar Bruna-Romero; Ricardo T. Gazzinelli; Mauricio M. Rodrigues

Interference or competition between CD8+ T cells restricted by distinct MHC-I molecules can be a powerful means to establish an immunodominant response. However, its importance during infections is still questionable. In this study, we describe that following infection of mice with the human pathogen Trypanosoma cruzi, an immunodominant CD8+ T cell immune response is developed directed to an H-2Kb-restricted epitope expressed by members of the trans-sialidase family of surface proteins. To determine whether this immunodominance was exerted over other non-H-2Kb-restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2Kb-, H-2Kk-, or H-2Kd-restricted CD8+ T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2Kb-restricted immunodominant epitope. In contrast, H-2Kk- or H-2Kd-restricted immune responses were significantly impaired in heterozygote infected mice when compared with homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi Ags. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8+ T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.


PLOS Pathogens | 2010

Platelet-Activating Factor Receptor Plays a Role in Lung Injury and Death Caused by Influenza A in Mice

Cristiana C. Garcia; Remo Castro Russo; Rodrigo Guabiraba; Caio T. Fagundes; Rafael B. Polidoro; Luciana P. Tavares; Ana Paula C. Salgado; Geovanni Dantas Cassali; Lirlândia P. Sousa; Alexandre V. Machado; Mauro M. Teixeira

Influenza A virus causes annual epidemics which affect millions of people worldwide. A recent Influenza pandemic brought new awareness over the health impact of the disease. It is thought that a severe inflammatory response against the virus contributes to disease severity and death. Therefore, modulating the effects of inflammatory mediators may represent a new therapy against Influenza infection. Platelet activating factor (PAF) receptor (PAFR) deficient mice were used to evaluate the role of the gene in a model of experimental infection with Influenza A/WSN/33 H1N1 or a reassortant Influenza A H3N1 subtype. The following parameters were evaluated: lethality, cell recruitment to the airways, lung pathology, viral titers and cytokine levels in lungs. The PAFR antagonist PCA4248 was also used after the onset of flu symptoms. Absence or antagonism of PAFR caused significant protection against flu-associated lethality and lung injury. Protection was correlated with decreased neutrophil recruitment, lung edema, vascular permeability and injury. There was no increase of viral load and greater recruitment of NK1.1+ cells. Antibody responses were similar in WT and PAFR-deficient mice and animals were protected from re-infection. Influenza infection induces the enzyme that synthesizes PAF, lyso-PAF acetyltransferase, an effect linked to activation of TLR7/8. Therefore, it is suggested that PAFR is a disease-associated gene and plays an important role in driving neutrophil influx and lung damage after infection of mice with two subtypes of Influenza A. Further studies should investigate whether targeting PAFR may be useful to reduce lung pathology associated with Influenza A virus infection in humans.


PLOS Pathogens | 2012

Pathogen-induced proapoptotic phenotype and high CD95 (Fas) expression accompany a suboptimal CD8+ T-cell response: reversal by adenoviral vaccine.

José Ronnie Vasconcelos; Oscar Bruna Romero; Adriano F. Araújo; Mariana R. Dominguez; Jonatan Ersching; Bruna Cunha de Alencar; Alexandre V. Machado; Ricardo T. Gazzinelli; Karina R. Bortoluci; Gustavo P. Amarante-Mendes; Marcela F. Lopes; Mauricio M. Rodrigues

MHC class Ia-restricted CD8+ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8+ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8+ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8+ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8+ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8+ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8+ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.


PLOS ONE | 2013

Complement C5 activation during influenza A infection in mice contributes to neutrophil recruitment and lung injury

Cristiana C. Garcia; Wynne Weston-Davies; Remo Castro Russo; Luciana P. Tavares; Milene Alvarenga Rachid; José C. Alves-Filho; Alexandre V. Machado; Bernhard Ryffel; Miles A. Nunn; Mauro M. Teixeira

Influenza virus A (IAV) causes annual epidemics and intermittent pandemics that affect millions of people worldwide. Potent inflammatory responses are commonly associated with severe cases of IAV infection. The complement system, an important mechanism of innate and humoral immune responses to infections, is activated during primary IAV infection and mediates, in association with natural IgM, viral neutralization by virion aggregation and coating of viral hemmagglutinin. Increased levels of the anaphylatoxin C5a were found in patients fatally infected with the most recent H1N1 pandemic virus. In this study, our aim was to evaluate whether targeting C5 activation alters inflammatory lung injury and viral load in a murine model of IAV infection. To address this question C57Bl/6j mice were infected intranasally with 104 PFU of the mouse adapted Influenza A virus A/WSN/33 (H1N1) or inoculated with PBS (Mock). We demonstrated that C5a is increased in bronchoalveolar lavage fluid (BALF) upon experimental IAV infection. To evaluate the role of C5, we used OmCI, a potent arthropod-derived inhibitor of C5 activation that binds to C5 and prevents release of C5a by complement. OmCI was given daily by intraperitoneal injection from the day of IAV infection until day 5. Treatment with OmCI only partially reduced C5a levels in BALF. However, there was significant inhibition of neutrophil and macrophage infiltration in the airways, Neutrophil Extracellular Traps (NETs) formation, death of leukocytes, lung epithelial injury and overall lung damage induced by the infection. There was no effect on viral load. Taken together, these data suggest that targeting C5 activation with OmCI during IAV infection could be a promising approach to reduce excessive inflammatory reactions associated with the severe forms of IAV infections.


Infection and Immunity | 2011

Heterologous Plasmid DNA Prime-Recombinant Human Adenovirus 5 Boost Vaccination Generates a Stable Pool of Protective Long-Lived CD8+ T Effector Memory Cells Specific for a Human Parasite, Trypanosoma cruzi

Paula Ordonhez Rigato; Bruna Cunha de Alencar; José Ronnie Vasconcelos; Mariana R. Dominguez; Adriano F. Araújo; Alexandre V. Machado; Ricardo T. Gazzinelli; Oscar Bruna-Romero; Mauricio M. Rodrigues

ABSTRACT Recently, we described a heterologous prime-boost strategy using plasmid DNA followed by replication-defective human recombinant adenovirus type 5 as a powerful strategy to elicit long-lived CD8+ T-cell-mediated protective immunity against experimental systemic infection of mice with a human intracellular protozoan parasite, Trypanosoma cruzi. In the present study, we further characterized the protective long-lived CD8+ T cells. We compared several functional and phenotypic aspects of specific CD8+ T cells present 14 or 98 days after the last immunizing dose and found the following: (i) the numbers of specific cells were similar, as determined by multimer staining or by determining the number of gamma interferon (IFN-γ)-secreting cells by enzyme-linked immunospot (ELISPOT) assay; (ii) these cells were equally cytotoxic in vivo; (iii) following in vitro stimulation, a slight decline in the frequency of multifunctional cells (CD107a+ IFN-γ+ or CD107a+ IFN-γ+ tumor necrosis factor alpha positive [TNF-α+]) was paralleled by a significant increase of CD107a singly positive cells after 98 days; (iv) the expression of several surface markers was identical, except for the reexpression of CD127 after 98 days; (v) the use of genetically deficient mice revealed a role for interleukin-12 (IL-12)/IL-23, but not IFN-γ, in the maintenance of these memory cells; and (vi) subsequent immunizations with an unrelated virus or a plasmid vaccine or the depletion of CD4+ T cells did not significantly erode the number or function of these CD8+ T cells during the 15-week period. From these results, we concluded that heterologous plasmid DNA prime-adenovirus boost vaccination generated a stable pool of functional protective long-lived CD8+ T cells with an effector memory phenotype.


PLOS ONE | 2011

Subdominant/cryptic CD8 T cell epitopes contribute to resistance against experimental infection with a human protozoan parasite.

Mariana R. Dominguez; Eduardo L. V. Silveira; José Ronnie Vasconcelos; Bruna Cunha de Alencar; Alexandre V. Machado; Oscar Bruna-Romero; Ricardo T. Gazzinelli; Mauricio M. Rodrigues

During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development.


PLOS Pathogens | 2015

A Human Type 5 Adenovirus-Based Trypanosoma cruzi Therapeutic Vaccine Re-programs Immune Response and Reverses Chronic Cardiomyopathy

Isabela Resende Pereira; Glaucia Vilar-Pereira; Virgínia Marques; Andrea Alice da Silva; Braulia Costa Caetano; Otacilio C. Moreira; Alexandre V. Machado; Oscar Bruna-Romero; Mauricio M. Rodrigues; Ricardo T. Gazzinelli; Joseli Lannes-Vieira

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ+ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas’ heart disease.


Vaccine | 2009

Strain-specific protective immunity following vaccination against experimental Trypanosoma cruzi infection.

Filipe A. Haolla; Carla Claser; Bruna Cunha de Alencar; Fanny Tzelepis; José Ronnie de Vasconcelos; Gabriel de Oliveira; Jaline Coutinho Silverio; Alexandre V. Machado; Joseli Lannes-Vieira; Oscar Bruna-Romero; Ricardo T. Gazzinelli; Ricardo Ribeiro dos Santos; Milena Botelho Pereira Soares; Mauricio M. Rodrigues

Immunisation with Amastigote Surface Protein 2 (asp-2) and trans-sialidase (ts) genes induces protective immunity in highly susceptible A/Sn mice, against infection with parasites of the Y strain of Trypanosoma cruzi. Based on immunological and biological strain variations in T. cruzi parasites, our goal was to validate our vaccination results using different parasite strains. Due to the importance of the CD8(+) T cells in protective immunity, we initially determined which strains expressed the immunodominant H-2K(k)-restricted epitope TEWETGQI. We tested eight strains, four of which elicited immune responses to this epitope (Y, G, Colombian and Colombia). We selected the Colombian and Colombia strains for our studies. A/Sn mice were immunised with different regimens using both T. cruzi genes (asp-2 and ts) simultaneously and subsequently challenged with blood trypomastigotes. Immune responses before the challenge were confirmed by the presence of specific antibodies and peptide-specific T cells. Genetic vaccination did not confer protective immunity against acute infection with a lethal dose of the Colombian strain. In contrast, we observed a drastic reduction in parasitemia and a significant increase in survival, following challenge with an otherwise lethal dose of the Colombia strain. In many surviving animals with late-stage chronic infection, we observed alterations in the hearts electrical conductivity, compared to naive mice. In summary, we concluded that immunity against T. cruzi antigens, similar to viruses and bacteria, may be strain-specific and have a negative impact on vaccine development.


Vaccine | 2012

Re-circulation of lymphocytes mediated by sphingosine-1-phosphate receptor-1 contributes to resistance against experimental infection with the protozoan parasite Trypanosoma cruzi

Mariana R. Dominguez; Jonatan Ersching; Ramon Lemos; Alexandre V. Machado; Oscar Bruna-Romero; Mauricio M. Rodrigues; José Ronnie Vasconcelos

T-cell mediated immune responses are critical for acquired immunity against infection by the intracellular protozoan parasite Trypanosoma cruzi. Despite its importance, it is currently unknown where protective T cells are primed and whether they need to re-circulate in order to exert their anti-parasitic effector functions. Here, we show that after subcutaneous challenge, CD11c(+)-dependent specific CD8(+) T-cell immune response to immunodominant parasite epitopes arises almost simultaneously in the draining lymph node (LN) and the spleen. However, until day 10 after infection, we observed a clear upregulation of activation markers only on the surface of CD11C(+)PDCA1(+) cells present in the LN and not in the spleen. Therefore, we hypothesized that CD8(+) T cells re-circulated rapidly from the LN to the spleen. We investigated this phenomenon by administering FTY720 to T. cruzi-infected mice to prevent egress of T cells from the LN by interfering specifically with signalling through sphingosine-1-phosphate receptor-1. In T. cruzi-infected mice receiving FTY720, CD8 T-cell immune responses were higher in the draining LN and significantly reduced in their spleen. Most importantly, FTY720 increased susceptibility to infection, as indicated by elevated parasitemia and accelerated mortality. Similarly, administration of FTY720 to mice genetically vaccinated with an immunodominant parasite antigen significantly reduced their protective immunity, as observed by the parasitemia and survival of vaccinated mice. We concluded that re-circulation of lymphocytes mediated by sphingosine-1-phosphate receptor-1 greatly contributes to acquired and vaccine-induced protective immunity against experimental infection with a human protozoan parasite.

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Ricardo T. Gazzinelli

Universidade Federal de Minas Gerais

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José Ronnie Vasconcelos

Federal University of São Paulo

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Mariana R. Dominguez

Federal University of São Paulo

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Bruna Cunha de Alencar

Federal University of São Paulo

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Braulia Costa Caetano

University of Massachusetts Medical School

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Adriano F. Araújo

Federal University of São Paulo

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Cristiana C. Garcia

Universidade Federal de Minas Gerais

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